Formation of intramolecular disulfide bridge

Oxidative folding of individual polymer chain was investigated for high molecular weight macromolecules by forming one intramolecular crosslink. Oxidation of the two inserted thiol groups into disulfide bond was explored to generate an intramolecular dynamic bridge and lead to chain cyclization. So far, several synthetic strategies have been described in literature toward oxidative thiol coupling by exploiting oxidizing agents such as metals, dimethyl sulfoxide (DMSO), ionic liquids, halogens, electrochemical oxidation or even by simple air exposure.²⁵⁰ DMSO and molecular oxygen (air-oxygen or oxygen pressure) has shown significant interests due to ease of handling and straightforward purification.²⁵¹ For both synthetic strategies, it has been reported that the oxidation of thiols into disulfide bonds are influenced by several external parameters, such as pH, temperature, solvent and thiols concentration.^252,253 Thiolate species are effectively able to form disulfide by simple air exposure while protonated thiols appear to be unreactive.²⁵² Therefore, thiol oxidations are strongly depending on the solution pH and can be promoted with the presence of base. Good yields have been described for reactions catalysed by amines.²⁵⁴ Moreover, thiol oxidation conducted in polar-aprotic solvents, such as dimethylformamide, is known to increase the oxidation rate of thiols to disulfide.^251,255

74 Oxidative single polymer chain folding was performed by using simultaneously air-oxygen and DMSO as oxidizing reagents according to a protocol adapted from the literature.¹⁷⁶ The linear polymer precursor l-poly(StyOH-co-MISH) was added dropwise via a syringe pump (48 h of addition) to a reaction mixture containing 5% vol. DMSO, 5% vol of the Hünig base N,N-diisopropylethylamine (DIPEA) and a large amount of DMF to avoid unwanted intermolecular cross-linking reactions (≈ 0.2 mg of polymer/mL after addition). The reaction was saturated with oxygen and was stirred for 6 days (Scheme 23) to afford the cyclic polymer locked via the formation of intramolecular disulfide bridge (c-poly(StyOH-co-MIS).

Scheme 23. Schematic illustration of the cyclization reaction induced by thiol oxidations into disulfide bridge in DMF with the presence of DMSO and base.

The cyclization reaction was monitored by SEC analysis in DMAc. Interestingly, no change in the SEC curves between the starting material and the resulting product could be observed (Figure 30A). The SEC traces could not give conclusive evidence of a hydrodynamic volume reduction, which would be obviously indicative of intramolecular cyclization. The formation of intermolecular crosslinking bonds is generally the main side reaction during single-chain polymer cyclization. However, di- tri- or multimerization was neither visible in the SEC traces. It should be mentioned that the reduction of hydrodynamic volume, caused by the formation of an intramolecular crosslink, scales inverse with the degree of polymerization of the polymer chain (DPn).²⁵⁶ In other words, the more the macromolecular chain is long, the less is the difference in the hydrodynamic volume exhibited by a linear precursor and its cyclic polymer analogue. In literature, polymers with DPn = 20-50 usually show a significant volume reduction in SEC measurements,^175,176 while large chains with DPn = 470 as applied in this study, were not expected to indicate a visible volume reduction by ring formation. Therefore, additional characterizations are obviously required to get clear indication on the resulting polymer topology after the cyclization reaction.

To gain insights into the oxidation of free thiols into disulfides, Ellman’s test was performed on the isolated cyclic polymer c-poly(StyOH-co-MIS). The experimental conditions

75 previously described for the Ellman’s test of the linear precursor, were used here to investigate the final thiol concentration. After adding DTNB compounds in excess to a solution containing c-poly(StyOH-co-MIS) in DMF, the solution remained colourless. This observation was confirmed by measuring the absorbance by UV-Vis spectroscopy. The spectrum demonstrated an absorbance of approximatively 0.02 at λ = 500 nm, indicating a nearly quantitative consumption of free thiol moieties (Figure 30B).

Figure 30. A) SEC traces of the polymers l-poly(StyOH-co-MISH) with free thiols (black solid line) and the resulting polymers c-poly(StyOH-co-MIS) after oxidation of the thiol group (dashed green line) B) UV-Vis spectrum resulting from the Ellman’s test on c-poly(StyOH-co-MIS) (green curve).

This result was encouraging, since it could suggest that thiol oxidation potentially occurred. However, it also appeared that the consumption of thiol did not reached completion.

This result was expectable considering the fact that a fraction of polymer chains could not be di-functionalized during the sequence-controlled copolymerization process. Statistically, it is plausible to assume that some macromolecules were mono-functionalized or tri-functionalized.

Indeed, the fraction of mono-functionalized chains could not be able to generate intramolecular disulfide bridge formation and oxidation of thiols to intramolecular disulfide bridge could not be quantitative. On the other hand, the fraction of tri-functionalized polymer chains could potentially form local disulfide bridge on one side of the macromolecules with a remaining free thiol on the other side.

It must be pointed out that the Ellman’s test on the linear precursor and cyclic polymer were performed to obtain a qualitative indication about the presence of thiols. The two obtained absorbance values were not quantitatively compared since both Ellman’s test were performed separately on the isolated linear precursor and the isolated cyclic polymer by using similar experimental conditions. Thus, a minor difference in the sample concentration could lead to misleading comparison of thiol concentration. Moreover, the Ellman’s test only indicated the potential disappearance of thiol functional groups and did not provide any information on the

76 resulting sulfur atom oxidation state. ¹H and 2D HSQC NMR spectroscopies were performed and could not give any evidence of -CH2-S- proton resonance shift, since the maleimide concentration was not sufficient to provide any signals even for the characterization of concentrated samples.

Due to limited amount of successful characterizations, no conclusion concerning the cyclization process could be determined at this stage. Hence, it appeared that the synthetic strategy developed in the current study, which consists in the transformation of the folded polymers into brush polymers to access AFM characterization, was highly required. Indeed, this situation clearly illustrated the high demand of additional characterization toward conformation analysis, for either complex macromolecular design or in this case, high molecular weight polymers. The transformation of the resulting polymer c-poly(StyOH-co-MIS) into brush polymers was performed for subsequent AFM microscopy characterization.

3.2.4. Synthesis of cyclic brush polymers

The transformation of the obtained polymers into brush polymers was targeted in the aim to gain insights into their morphology by AFM analysis. Previously, it was shown that disulfide bridges are chemically stable during controlled radical polymerization and do not interfere in the polymerization process. Hence, the «grafting from» approach which relies on the preparation of bottlebrush polymers by growing polymer side chains via CRP polymerization on a polymeric backbone,²²⁵ was selected in this study. This method is more convenient for subsequent AFM characterization due to achievable high grafting densities and straightforward bottlebrush purification by filtration and precipitation. So far, grafting from reactions have been mostly conducted from polyethylene,²⁵⁷ polyvinylchloride,²⁵⁸ polyisobutylene²⁵⁹ or even poly(meth)acrylic backbones.³⁸ To the best of our knowledge, the synthesis of densely brush macromolecules having a poly(styrene) backbone and poly(n-butyl acrylate) side chains by using the «grafting from» approach was not described in literature. Thus, a prior study was performed to investigate the controlled and successful preparation of linear brush polymers composed of such chemical structure.

3.2.4.1. Linear brush polymer synthesis via « grafting from approach »

The «grafting from» method allows the preparation of bottlebrushes with high grafting density and significant uniformity between macromolecules. The first requirement for a successful "grafting from" reaction is a preformed polymer backbone with distributed initiating groups for subsequent polymerization of side chains. So far, grafting side chains has been mainly achieved by performing ATRP process initiated by pendant α-bromoester groups on

77 poly(methacrylate) backbones.²²⁶ In this section, the synthesis of brush polymers composed of styrenic backbone and n-butyl acrylate side chains was studied by using the «grafting from»

approach. Synthesis of poly(tert-butoxystyrene) followed by a tert-butyl deprotection reaction afforded a functional polystyrene backbone. Then, polymer post-modification was implemented to introduce initiator groups on the styrenic backbone and ATRP polymerization was subsequently conducted.

A linear poly(tert-butoxystyrene) precursor , with a DPn in the range of 380 monomer units (l-poly(StyOtBu)380), was prepared by NMP polymerization by using the novel bifunctional initiator (Bis-BB-SG1). The obtained homopolymer was successfully characterized by ¹H NMR spectroscopy and SEC analysis (SEC in DMAc, Mn,app = 66000 and Ð = 1.16, see section 6.3.13). The following step consisted in the removal of tert-butyl groups present on the phenolic backbone. Tert-butyl deprotection was achieved by hydrolysis with hydrochloric acid (HCl) in dioxane at high temperature to afford the linear poly(4-hydroxystyrene) precursor (l-poly(StyOH)380). ¹H NMR spectroscopy indicated a full deprotection of the 4-hydroxystyrene units, since the peak corresponding to the resonances of tert-butyl groups fully disappeared. The SEC analysis evidenced the formation of macromolecules with controlled molecular weight and narrow molecular weight distribution (SEC in DMAc, Mn, app = 85000, and Ð = 1.23, see section 6.3.13).

The next step consisted in the introduction of α-bromoester groups on the poly(4-hydroxystyrene) backbone which are necessary for the subsequent ATRP polymerization. For this purpose, the hydroxyl functionalities of the linear homopolymer l-poly(StyOH)380 were esterified by treatment with bromopropionyl bromide in the presence of pyridine to yield the poly(styrene) backbone bearing pendant α-bromoester segments (l-poly(StyBr)380) (Scheme 24). The resulting ATRP macroinitiator was characterized by ¹H NMR and SEC analysis. The

1H NMR spectrum showed the appearance of new signals at 4.58 and 1.94 ppm corresponding to the introduced CH-Br and CH3-CH side groups, respectively (see section 6.4, Figure 91).

Furthermore, by comparing the integration of the peak areas of the new formed signals with those of aromatic protons from the poly(styrene), a nearly quantitative conversion of hydroxyl group into bromoester was estimated (97% of conversion). SEC characterization demonstrated a shift to higher molecular weight region compared to the phenolic backbone precursor and indicated Mn, app = 88000 with Ð = 1.24 (SEC trace shown in Figure 31).

78 Scheme 24. Synthesis of the linear ATRP-macroinitiator (l-poly(StyBr)380). Esterification of the hydroxyl groups of l-poly(StyOH)380 with bromopropionyl bromide in the presence of pyridine at room temperature.

After the successful preparation of the poly(styrene) derivative macroinitiator, ATRP polymerization of n-butyl acrylate monomer could be implemented to grow polymer side chains on the macromolecules. It must be mentioned that polymerization on macroinitiators requires generally different experimental conditions than a traditional ATRP using low molecular weight monofunctional initiator. Due to a high local concentration of initiation sites of the polymer backbone, termination reactions such as radical-radical coupling can occur more likely during the controlled radical polymerization of side chains. To avoid these unwanted reactions, the polymerization of side chains are performed under dilute conditions. More precisely, the desired length of the side chains, is obtained by conducting the ATRP process in a large excess of monomer and subsequently stopping the reaction at low monomer conversions, usually around 10%.³⁸ Moreover, using a sub-stoichiometric amount of cuprous catalyst and adding a ~5% of copper (II) bromide (Cu^IIBr2)reversibly deactivate the growing polymer chains and thus suppress termination reactions.³⁸ In this study, ATRP of n-butyl acrylate was conducted at 80°C in ethyl methyl ketone, by using a small amount of CuBr2, the catalytic complex CuBr/N,N,N′,N′′,N′′-pentamethyl diethylenetriamine (PMDETA) and l-poly(Sty-Br)380 as macroinitiator, with a ratio of [Br: CuBr: PMDETA: CuBr2: nBuA] = [1: 0.5: 0.525: 0.025: 500] (Scheme 25). The monomer conversion was monitored by ¹H NMR spectroscopy and the polymerization was stopped at approximatively 5% of conversion after 1.5 h. The average degree of polymerization for the poly(nBuA) side chains was approximatively of DPn = 25 calculated from monomer conversion data and similar estimation was obtained from gravimetric method (DPn = 22).

79 Scheme 25. Synthesis of linear brush polymer l-poly(Sty)380-g-poly(nBuA)25. ATRP polymerization of n-butyl acrylate initiated by the macroinitiator l-poly(StyBr)380 in ethyl methyl ketone at 80°C with the ratio of reagents of [Br: CuBr: PMDETA: CuBr2: nBuA] = [1: 0.5: 0.525: 0.025: 500].

The resulting linear brush polymer l-poly(Sty)380-g-poly(nBuA)25 was analysed by proton NMR and SEC chromatography. ¹H NMR spectrum indicated the appearance of new signals corresponding to the n-butyl acrylate units while the signals corresponding to the styrenic backbone remained unchanged (Figure 92). SEC traces of the brush copolymer clearly evidenced a shift toward lower elution volume, which is qualitatively indicative of an increase of molecular weight after ATRP polymerization (Figure 31, Mn, app = 517000 and Ð = 1.28).

It must be pointed out that the molecular weight indicated by SEC analysis was different than the molecular weight estimated by gravimetry or by monomer conversion from proton NMR.

However, this observation was expectable since SEC data analysis using refractive index detection is calibrated vs. linear polystyrene standards and does not yield accurate molecular weight data.³⁸ In fact, due to the high compact structure of densely grafted bottlebrush polymers, the molecular weight to hydrodynamic volume relation strongly differs from linear polystyrene used for SEC calibration. Besides, the polydispersity of the brush is merely determined by the polydispersity of the backbone.^39,260 In this case, after the polymerization of side chains, the polydispersity nearly remained unchanged, indicating the good control of the n-butyl acrylate ATRP process. Using a high ratio of monomer to initiator and stopping the polymerization at low conversion avoided successfully the undesirable side reactions and enabled the preparation of the desired brushes with poly(nBuA)25 side chains.

80 Figure 31. SEC traces of the macroinitiator precursor polymer l-poly(StyBr)380 (orange curve) and the resulting linear bottlebrush polymer l-poly(Sty)380-g-poly(nBuA)25 (blue curve).

AFM microscopy was used to visualize the molecular morphology of the resulting linear bottlebrush polymer l-poly(Sty)380-g-poly(nBuA)25. For AFM studies, the sample was prepared by spin-coating a dilute solution of crude polymer in chloroform (ca. 0.01 mg/mL) on a freshly cleaved mica substrate to obtain a monomolecular film.

Figure 32 shows the obtained height and amplitude images. In both, bottlebrush polymers were clearly observed and as expected, exhibited worm-like molecular morphology.

To further demonstrate the successful preparation of bottlebrush macromolecules with high grafting density, the backbone contraction was evaluated. Statistical molecular dimensions were determined by direct measurement of a significant ensemble of thirty bottlebrush macromolecules. The statistical average contour length, uncorrected full width at half maximum (FWHM) and height were estimated in the range of 98 ± 15 nm, 15 ± 3 nm, and 1.2

± 0.2 nm, respectively. The statistical average contour length is very close to the theoretical maximal length estimated for polymer backbone of DPn = 380 with fully extended all-trans repeated unit bond conformations (lunit, max = 0.24 nm) which is in the range of 91 nm (380 × 0.24 nm). Hence, in this case, it appeared that the bottlebrush morphology was fully extended, which indirectly suggested the high grafting density of poly(n-butyl acrylate) side chains on the styrenic backbone.

This investigation showed that the «grafting from» method led to well-controlled synthesis of bottlebrush polymers composed of poly(styrene) backbone and poly(n-butyl acrylate) side chains. Thus, this straightforward synthetic strategy seemed to be a promising pathway to transform cyclic polymers (previously cyclized by intramolecular disulfide bridge), into cyclic brush polymers in order to allow AFM microscopy characterization.

81 Figure 32. AFM micrograph of the linear bottlebrush polymer l-poly(Sty)380-g-poly(nBuA)25. A) Height image. B) Amplitude image.

3.2.4.2. Folded brush polymers synthesis

Previously, attempts toward single polymer chain compaction were conducted on high molecular weight macromolecules by forming one single intramolecular disulfide bridge (See section 3.2.3). However, standard characterization tools usually utilized to evidence cyclic polymer topology, such as SEC chromatography or NMR spectroscopy, could not prove in this case the cyclization process. In the aim to visualize directly the resulting polymer topology by AFM microscopy, the potential cyclic macromolecules were transformed into brush polymers.

The grafting from approach was performed following the reaction conditions discussed in the previous section (see section 3.2.4.1). Pendant α-bromoester fragments were introduced on the polymer backbone c-poly(StyOH-co-MIS) by esterification of the hydroxyl units with bromopropionyl bromide in the presence of pyridine to yield the poly(styrene) macroinitiator with pendant α-bromoester segments (c-poly(StyBr-co-MIS)). The polymer was characterized by ¹H NMR (Figure 33). New signals appeared at 4.58 and 1.94 ppm, corresponding to the proton resonances located on the bromoester fragments. Nearly all the 4-hydroxystyrene backbone units were successfully transformed into initiating sites for subsequent ATRP polymerization (98% estimated by ¹H NMR). Furthermore, the macroinitiator was analysed by SEC chromatography in DMAc, which demonstrated a shift of the main peak toward higher molecular weight region and an unchanged molecular weight distribution (Mn, app = 92000 and Đ = 1.30, SEC trace in Figure 34).

The obtained poly(styrene) derivative macroinitiator was then used to implement ATRP polymerization of n-butyl acrylate monomer and grow polymer side chains on the macromolecules. For this purpose, ATRP of n-butyl acrylate was conducted at 80 °C in ethyl

82 methyl ketone, by using a small amount of CuBr2, the catalytic complex CuBr/N,N,N′,N′′,N′′-pentamethyl diethylenetriamine (PMDETA) and c-poly(StyBr-co-MIS) as macroinitiator, with a ratio of [Br: CuBr: PMDETA: CuBr2: nBuA] = [1: 0.5: 0.525: 0.025: 500]. The monomer conversion was monitored by ¹H NMR spectroscopy and the polymerization was stopped at

~9% of conversion after 2 h, to afford the brush polymer (c-poly(Sty)470-g-poly(nBuA)40).

Figure 33. ¹H NMR spectrum of the obtained macroinitiator c-poly(StyBr-co-MIS) in CDCl3 after esterification of the 4-hydroxystyrene backbone units by bromopropionyl bromide in presence of pyridine.

An average degree of polymerization for the poly(nBuA) side chains was estimated of DPn = 40 units, calculated from monomer conversion data by NMR (DPn, NMR = 45) and gravimetry (DPn, grav = 35). The isolated cyclic brush macromolecule was characterized by proton spectroscopy and SEC chromatography. In the NMR spectrum, signals corresponding to n-butyl acrylate resonances appeared at 4.03, 1.90 - 1.38 and 0.92 ppm while signals corresponding to the proton resonances of backbone styrene units could still be observed at 7.03 – 5.81 ppm (see section 6.4, Figure 96). SEC chromatography indicated a peak shifted to lower elution time, demonstrating an obvious increase in molecular weight after the ATRP polymerization and a quantitative transformation of the macroinitiator into brush polymer (Figure 34). However, the elugram revealed a polymer peak with a significant shoulder at the high molecular weight flank and a broadening at the low molecular weight flank, which increased the molecular weight distribution (Mn, app = 465000 and Ð = 1.43). The measured Mn, app of the shoulder on the left-hand side is approximatively the double of the Mn, app

83 exhibited by the main peak (Mn, app (shoulder) = 916000 vs Mn, app (main peak) = 451000). These three different populations of polymer could potentially correspond to polymer chain dimers (high molecular weight shoulder), linear polymers (main peak) chain and the desired cyclic polymers (broadening at the low molecular weight flank). As mentioned previously, cyclic polymers exhibit smaller hydrodynamic volume compared to their linear analogue. This peak interpretation would indicate a poor cyclization yield while linear analogue polymers and linear polymer chain dimers could be the main macromolecular populations. Nevertheless, it remained difficult to get precise and clear information either on the topology of the obtained polymers or on the cyclization yield, with this conventional analytical technique.

Deconvolution of SEC peak was tested in the aim to quantify each polymer populations.

However, due to the excessive coelution of the main linear polymers with the two other populations (high and low molecular weight shoulder), the estimation could be obviously misled (data not shown).

Figure 34. SEC traces of the macroinitiator c-poly(StyBr-co-MIS) (black curve) and the resulting cyclic bottlebrush polymers c-poly(Sty)470-g-poly(nBuA)40 (green curve) after ATRP polymerization of n-butyl acrylate monomers on the macroinitiator

Besides, it appeared that the high molecular weight peak shoulder observed in the elugram, which corresponded potentially to polymer chain dimers, increased considerably during the brush polymer synthesis step. Thus, this observation strongly indicated that side reactions probably occurred during the CRP process. Such chain dimerization of brush polymers could arise from termination reactions, such as radical-radical coupling. Indeed, in this case, the ATRP process was stopped at higher conversion of monomer (~10%), which could potentially cause radical coupling side reactions and could explain the significant increase of higher molecular weight brush polymers. Nevertheless, the obtained brush polymer was subsequently characterized by AFM microscopy to gain insight into the polymer topologies of the overall sample.

84 3.2.5. Macromolecular imaging of cyclic brush polymers

The folded polymers have been transformed into folded molecular brushes to allow conformation analysis by Atomic Force Microscopy. Indeed, while SEC analysis could only indicate the presence of three distinct polymer populations in the overall sample, AFM

The folded polymers have been transformed into folded molecular brushes to allow conformation analysis by Atomic Force Microscopy. Indeed, while SEC analysis could only indicate the presence of three distinct polymer populations in the overall sample, AFM